Simultaneous chemical separation and surface-enhanced Raman spectral detection using metal-doped sol-gels

ABSTRACT

Sol-gel beds and deposits are utilized for SERS analysis of liquid analytes. The use of the same medium to both separate the chemicals and also for SERS greatly reduces the complexity of such apparatus and enhances the efficiency of the method.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

The United States Government has rights in this invention pursuant toNational Science Foundation Contract No. DMI-0060258.

BACKGROUND OF THE INVENTION

The combination of chemical separation and analysis has long beenrecognized as invaluable to the analytical chemist in identifyingchemicals at extremely low concentrations in complex matrices. Forexample, a drug and its metabolites can be effectively separated fromblood plasma, using gas chromatography, and thereafter identified by thechemical fragments detected by mass spectrometry (see J. Chamberlain,The Analysis of Drugs in Biological Fluids, CRC Press, Boca Raton, 995,2nd ed. chap. 6 and 7).

More recently, the combination of liquid chromatography, or flowinjection analysis, with surface-enhanced Raman spectroscopy (SERS) hasbeen investigated for such applications (see J-M. L. Sequaris and E.Koglin, Anal. Chem., 59,525 (1987); R. D. Freeman, R. M. Hanmaker, C. E.Meloan, and W. G. Fateley, Appl. Spectrosc., 42,456-460 (1988); F. Ni,R. Sheng and T. M. Cotton, Anal. Chem., 62, 1958(1990); G. T. Taylor, S.K. Shanna and K. Mohanan, Appl. Spectrosc., 44,635 (1990); R. Sheng, F.Ni and T. M. Cotton, Anal. Chem., 63,437 (1991); N. J. Pothier and R. K.Force. Appl. Spectrosc., 46, 147 (1992); L. M. Cabalin, A. Ruperez andJ. J. Laserna, Talanta, 40, 1741 (1993); K. T. Carron and B. J. Kennedy,Anal. Chem., 67. 3353 (1995); L. M. Cabalin, A. Ruperez and J. J.Laserna, Anal. Chim. Acta, 318, 203(1996); N. J. Szabo and I. D.Winefordner, Appl. Spectrosc., 51.965 (1997); B. J. Kennedy, R. Milofskyand K. T. Carron, Anal. Chem., 69, 4708 (1997); and W. F. Nirode, G. L.Devault, M. J. Sepaniak and R. O. Cole, Anal. Chem., 72, 1866(2000)).Advantages of this combination of techniques include minimal samplepreparation requirements, unrestricted use of water in the mobile phase,high chemical specificity through abundant molecular vibrationalinformation, and extreme sensitivity, as demonstrated by the detectionof single molecules. (See K. Kneipp, Y. Wang, R. R. Dasari and M. S.Feld, Appl. Spectrosc., 49,780(1995); and S. Nie and S. R. Emory,Science, 275, 1102 (1997)).

Previous research has employed primarily the three most common methodsof generating SERS; i.e., using roughened silver or gold electrodes,using silver or gold-coated substrates, and using silver or goldcolloids for detecting separated analytes. The lattermost method hasgained the greatest amount of attention, since colloids can be preparedeasily and inexpensively, and mixing of the colloids with thechromatographic column effluent, using flow injection, is reproducible.Care must be taken however to control aggregation of the colloids sothat the amount of Raman signal enhancement is maintained. Also, a rangeof experimental variables, such as analyte concentration and pH, canstrongly influence aggregation and, to some extent, limit applications;the choice of mobile phase is similarly limited by the need to maintaincolloid integrity.

Recently, as described by Farquharson et al. in copending and commonlyowned U.S. application Ser. No. 09/704,818 (published as InternationalPublication No. WO 01/33189 A2, dated 10 May 2001), the entirespecification of which is hereby incorporated by reference thereto,sol-gels have been developed to trap silver or gold particles as animproved method of generating plasmons for SERS (see also S.Farquharson, P. Maksymiuk, K. Ong and S. D. Christesen, SPIE, 4577,166(2002); F. Akbarian, B. S. Dunn and J. I. Zink, J. Chem. Phys., 99,3892 (1995); T. Murphy, H. Schmidt and H. D. Kronfeldt. SPIE, 3105, 40(1997); and Y. Lee, S. Dai and J. Young, J. Raman Spectrosc. 28, 635(1997)). It is appreciated that, once the sol-gel has formed, theparticle size and aggregation of the metal dopant are stabilized, albeitchanges in pH may still result in variable Raman signal intensities,such as in the case of weak acids and bases, where the relativeconcentrations of the ionized and unionized forms may be influenced.Also, it has been shown that many of the common solvents, such asacetone, methanol, and water, can be used equally with these metal-dopedsol-gels in generating SERS of analytes.

In accordance with other recent developments, moreover, sol-gels havebeen used as the stationary phase in columns for liquid- and gas-phasechromatography, affording advantages in both the preparation of columnsand also in their performance. The sol-gel approach enablesdeactivation, coating, and immobilization to be combined as a singlestep, while the sol-gels have shown reduced tailing, improvedseparation, and broader application to solvents and analytes.

Microchip devices have also been employed for effecting chemicalseparations (see Jacobson, S. C., Hergenröder, R., Koutny, L. B., &Ramsey, J. M. “High-Speed Separations on a Microchip,” Anal. Chem., 66,1114-1118 (1994); Jacobson, S. C., Hergenröder, R., Koutny, L. B.,Warmack, R. J., & Ramsey, J. M. “Effects of Injection Schemes and ColumnGeometry on the Performance of Microchip Electrophoreis Devices,” Anal.Chem., 66, 1107-1113 (1994); Jacobson, S. C. Hergenröder, R., Koutny, L.B. & Ramsey, J. M. “Open Channel Electro-chromatography on a Microchip,”Anal. Chem., 66, 2369-2373 (1994); and Moore, Jr., A. W., Jacobson, S.C. & Ramsey, J. M. “Microchip Separations of Neutral Species viaMicellar Electrokinetic Capillary Chromatography,” Anal. Chem., 67,4184-4189 (1995)).

SUMMARY OF THE INVENTION

It is the broad object of the present invention to provide a novelmethod and apparatus for the separation and immediate qualitative andquantitative analysis of components of liquid samples.

It has now been found that certain of the foregoing and related objectsof the invention are attained by the provision of a method forsubstantially simultaneously separating and detecting at least oneanalyte compound, wherein a sample solution containing a plurality ofcompounds, including at least one analyte compound, is transportedthrough or along a metal-doped, surface-enhanced Raman-active sol-gelmedium in sufficiently intimate chemical and/or physical contact foreffecting separation of the “at least one” analyte compound.Substantially concurrently, the medium is irradiated with excitationradiation to generate inelastically scattered Raman radiation, at leasta portion of which is collected and analyzed to determine the presenceof the analyte compound in the sample solution. The sol-gel medium willusually comprise or define an elongate path for the sample solution,such as in a capillary column or a microchip channel.

Other objects of the invention are attained by the provision ofapparatus for effecting, substantially simultaneously, separation of atleast one analyte compound from a sample solution containing a pluralityof dissolved compounds, and detection of the “at least one” analytecompound. The apparatus comprises elongate containment means forcontaining a porous medium and having an entrance for introducing asample solution thereinto, and a quantity of at least one metal-doped,surface-enhanced Raman-active sol-gel contained in the containment meansand providing such a porous medium. The containment means issufficiently transparent to excitation radiation, at least at onelocation spaced from the entrance along its length, to permittransmission of excitation radiation effective for generating measurableamounts of inelastically scattered Raman radiation, and it issufficiently transparent to such scattered radiation, at least at thatlocation, to permit transmission of measurable amounts thereof. Theporous medium defines a flow path through the containment means, past atleast the “one location,” and is of such character as to promoteintimate physical and/or chemical contact with a sample solutiontransported along the defined flow path.

The apparatus of the invention may desirably include a microchip cardsubstrate, with the elongate containment means comprising a microchannelin the substrate or a capillary tube on the substrate, and with thesubstrate having a plurality of ports communicating with the channel andproviding entrance-defining and exit-defining means. The porous mediumwill, in such embodiments, advantageously comprise a lining deposited ona wall of the channel or tube, or a packed bed in the channel or tube,defining the sample flow path.

The present invention uniquely combines two functions of sol-gels; i.e.,(1) the ability to separate chemicals, and (2) the ability to immobilizemetal particles that promote SER scattered radiation from chemicals insolution, which in turn enables analyses to be performed in a highlyeffective and efficient manner. One or more suitable optical probes,capable of excitation and collection of Raman photons, monitors the onelocation of the transparent column for the detection of separatedchemical species, thereby enabling a complete analysis to beaccomplished. The rate of the chemical and physical contact that isnecessary for effecting separation of the species can be promoted bydriving the analyte solution through or along a sol-gel bed or depositunder applied positive or negative pressure.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagrammatic representation of a packed bed column used forseparation and analysis of dissolved analytes, showing both thetraditional, gravity-flow (with inherent capillary action) method ofsolution transport, with a single sampling point, and also avacuum-assisted transport method with multiple sampling points;

FIG. 2 is a plot of Raman band relative intensity over a period of 100minutes, constituting an elution profile of phenyl acetylene (PA) andp-amino-benzoic acid (PABA);

FIG. 3 presents a series of spectra, taken at five points along thelength of a sol-gel packed column used for separation and measurement ofconcentrations of PA and PABA; and

FIG. 4 is a diagrammatic representation of a microchip deviceincorporating a SER-active sol-gel chemical separation channel andenabled by the present invention.

DETAILED DESCRIPTION OF THE PREFERRED AND ILLUSTRATED EMBODIMENTS

The silver-doped SER-active sol-gels employed in the examples thatfollow were prepared in accordance with the method of Lee andFarquharson (SPIE 4206, 140 (2001). In essence, a silver amine complex,consisting of a 5:1 v/v solution of 1 N AgNO₃ and 28% NH₃OH, is mixedwith an alkoxide, consisting of a 2:1 v/v solution of methanol andtetramethyl orthosilicate (TMOS) in a 1:8 v/v silver amine:alkoxideratio.

As an example of a fabrication technique that can be used in thepractice of the invention, a 0.15 mL aliquot of the foregoing mixture istransferred to a 2 mL glass vial, which is spun to coat its insidewalls. After sol-gel formation, the incorporated silver ions are reducedwith dilute sodium borohydride, followed by a water wash to removeresidual reducing agent. The sol-gel coating is scraped from the wallsof the vial, and is converted to a homogeneous powder by grinding with amortar and pestle.

As depicted in FIG. 1, the ground sol-gel 10 is packed into a 5 mmsegment of a 4 cm length of a 1.0 mm diameter melting point capillarytube 12, using a sterile cotton plug 14 to hold the powder in place, andthe top is fit with a 1.0 mL disposable plastic pipette (not shown) toallow delivery of 10 μL samples to the rudimentary liquid chromatographycolumn so prepared. A diaphragm pump (also not shown) is attached to theexit end of the column to enable vacuum-assisted transport of the testsolution through the sol-gel bed.

The column is fixed vertically at the focal point of a microscopeobjective (20×0.4) attached to an XYZ positioning stage, to focus thebeam into the sample and to collect radiation scattered back along theaxis of incidence. A notch filter is provided to reflect the excitationlaser beam to the microscope objective, and to pass the collectedRaman-scattered radiation.

Two 3 m lengths of fiber optic were used to deliver the laser energy(200 micron diameter) and to collect the Raman radiation (365 microndiameter). A Nd:YAG laser provided 50 mW of 1064 nm excitation radiationat the sample, and a Fourier transform Raman spectrometer, equipped withan InGaAs detector, was used for spectra acquisition.

EXAMPLE ONE

Insofar as the flow of analyte solution is concerned, the followingexperiment (depicted along the left side of FIG. 1) mimics traditionalliquid chromatography. A solution of 8×10⁻³M p-aminobenzoic acid and4×10⁻³M phenyl acetylene was prepared in methanol to demonstrateseparation of polar and non-polar chemicals. A 10 μl quantity of thesolution was added to the top of a separation and analysis column,constituted and assembled as hereinabove described. A 1 mL quantity ofmethanol was added as a carrier solvent, and allowed to elute under theforces of gravity and capillary action only. Using an optical probecoupled to a Raman spectrometer, which measured the surface-enhancedRaman spectra at the bottom of the column as a function of time, it wasconfirmed that the methanol solvent caries the non-polar PA through thecolumn ahead of the polar PABA.

More specifically the microscope objective was positioned 0.5 mm fromthe bottom edge of the 5 mm length of packed sol-gel, and scans weremade and averaged every 30 seconds to produce spectra. Unique bands forPA and PABA, at 1985 cm⁻¹ and 850 cm⁻¹, respectively, were used to plotrelative concentration as a function of time. FIG. 2 shows the elutionprofiles generated for both analytes during a 100 minute test period,which verify that chemical separation does occur. These data also showhowever that, in the absence of any external driving force, asignificant period of time is required.

EXAMPLE TWO

This example demonstrates that techniques can be applied for driving thesolution through the column to substantially reduce analysis time. Thus,a second experiment (depicted along the right side of FIG. 1) employs anidentical sample but uses a 50/50 v/v mixture of methanol and water asthe carrier solvent, rather than methanol alone. In addition, a vacuumof a 50 cm of Hg was applied for 30 seconds to draw the sample throughthe column. Due to the addition of water in the solvent, the separationis reversed because, in the present case, the alkoxide, TMOS, used toprepare the sol-gel is hydrophilic (i.e., water carries the polar PABAthrough the column first), demonstrating flexibility of the concept.

Since the entire length of the column is SER-active, moreover, theextent of separation could be measured by moving the microscopeobjective to five different positions along its length, enabling thecollection of spectra at each level. More specifically, spectra, plottedin FIG. 3, were collected at five discrete points spaced 1-mm apart, thefirst being located at a level 0.5 mm from the top edge of the sol-gelbed, with each spectrum consisting of scans averaged for 30 seconds.Spectra (1) and (5), obtained at the top and bottom of the column,indicate pure PA and PABA, respectively; the intermediate spectrarepresent mixtures of the two analytes.

Because there was no need to wait for the analytes to elute past asingle measurement point at the end of the column (i.e., the separatedchemicals can be measured wherever they occur along the column, since itis SER-active along its entire length), each analyte could be identifiedquickly; complete analysis was performed in three minutes, as comparedto at least 80 minutes in the traditional method. The time savingsrealized provides many significant benefits, particularly for tracechemical analyses of multi-component systems.

The series of spectra presented in FIG. 3 also demonstrates the power ofRaman spectroscopy, in that each chemical can be easily identified,either isolated or as a mixture. Although previous knowledge of, orexpectation as to, the sample composition simplifies the task, spectralmatching and deconvolution software programs, or like techniques, can beused to handle unknown components.

EXAMPLE THREE

A microchip chemical analyzer, diagrammatically illustrated in FIG. 4,constitutes a form of apparatus enabled by the present invention. Theanalyzer comprises a card, generally designated by the numeral 20, whichcontains a sample input port 21, a solvent entry channel 22, valves 24,25, 26 and 27, and a SER-active sol-gel microchannel 28. In thisinstance the sol-gel takes the form of a porous lining deposited on thewall of the channel 28 (albeit a packed channel is also feasible), andan applied vacuum driving force promotes rapid passage through thechannel 28 coupled with the physical and chemical contact required foreffective separation.

In use, a sample (e.g., a drop of blood) is applied to a porous cover,such as a membrane or sponge overlying the sample entry port 21 (or theport may be of septum-like form), typically using an eye dropper, apipette, or a syringe. The sample is then urged, such as by vacuumapplied at the waste chamber 35 (or alternatively, by positive pressuresuch as may be applied by a pipette or syringe, always using of courseappropriate connections), into a load channel section 30, for whichpurpose the valves 24 and 25 would be opened and the valves 26 and 27would be closed. Then, with valves 24 and 25 closed to isolate thesample entry port 21 and the waste chamber 35, and the valves 26 and 27opened, solvent is drawn (or alternatively, pushed) through the channel22 to drive the loaded sample through the passage of the channel 28 towaste chamber 36, again with vacuum (or pressure) applied thereat.Chemical components of the sample interact with the sol-gel deposit andare thereby separated, allowing identification and quantitation of thetarget analyte(s) by SER spectroscopy using a Raman optical probe suchas a suitably mounted objective 34 with the appropriate interfaceoptics. The microchip card 20 would typically fit onto a platform thataligns interconnects for the sample/solvent delivery and flow-controlsystem, and that dynamically positions the Raman probe objective toenable spectral analyses to be effected along the length of theSERS-active portion of the channel 28, as described.

It will be appreciated that virtually any sol-gel, in powdered,particulate or other finely divided form, or in the form of a porous,passage-defining deposit, can be used as a separation/analysis medium inthe practice of the present invention. Selectivity may be afforded bythe inherent electro-potential of the metal dopant (electronegative orelectropositive), by the hydrophobic or hydrophilic nature of thesol-gel medium, etc. Thus, while the examples set forth above employ asilver-doped sol-gel, doping with gold is regarded to be equallyimportant; copper, and less desirably nickel, palladium, and platinum,and alloys and mixtures thereof, can of course be utilized as well.

The nature and structure of the containment “vessel” can vary widely,and is not limited to columns; as described above, for example, theanalysis apparatus may comprise glass or plastic microchannelsincorporated into microchip analyzers. Albeit the sample path willusually be rectilinear, it will be appreciated that the elongate pathreferred to herein may be curvilinear and of relatively complex,compound configuration as well. A fluidic device used to add solvent andpush and/or pull the sample through the SER-active medium, for effectingsample introduction and separation, can take many different forms, itbeing appreciated that the functional features of the device may beimportant from the standpoint of assuring the intimacy of contactnecessary for efficient separation of the analyte compound(s).Similarly, the Raman probe can take many different forms, as will beapparent to those skilled in the art; as one example, however, the probemay desirably comprise six collection optical fibers surrounding oneexcitation fiber.

Numerous applications can benefit from the method and apparatus of theinvention, including, for example, the detection of contaminants ingroundwater (e.g. CN⁻, CrO₄ ⁻), the determination of drug presence andefficacy, by analysis for a parent constituent and/or its metabolites ina biological fluid, and the detection of chemical agent hydrolysisproducts in poisoned water; other applications will readily occur tothose skilled in the art.

It should be understood that the term “solution” is used in a broadsense in the present description and claims. It is intended to encompasscolloidal suspensions (of dispersed solid, semisolid, and liquidparticles) in a fluid (gas or liquid) continuous phase, as well as truesolutions (i.e., at the molecular or ionic level) of one or moredissolved substances in a simple or mixed fluid solvent.

Thus, it can be seen that the present invention provides a method andapparatus by which a metal-doped sol-gel can be used for thesubstantially simultaneous separation and SERS analysis of chemicals insolution.

1. A method for substantially simultaneously separating and detecting atleast one analyte compound, comprising: providing a sample solutioncontaining a plurality of compounds, including at least one analytecompound; providing containment means that is sufficiently transparentto excitation radiation, at least at one location, to permittransmission of excitation radiation effective for generating measurableamounts of surface enhanced Raman scattered radiation, and that issufficiently transparent to such scattered Raman radiation, at least atsaid one location, to permit transmission of measurable amounts of suchscattered Raman radiation, said containment means containing ametal-doped, surface-enhanced Raman-active sol-gel medium; transportinginto said containment means a quantity of said sample solution, with adriving force applied to promote flow, to thereby distribute said samplesolution upon said sol-gel medium in sufficiently intimate contact foreffecting separation of said at least one analyte compound;substantially concurrently irradiating said medium, and sample solutiondistributed thereon, with excitation radiation, at least at said onelocation, to generate surface enhanced Raman scattered radiation;collecting at least a portion of said scattered Raman radiation; andanalyzing said collected radiation to determine the essence of theanalyte compound in said sample solution.
 2. The method of claim 1wherein said sol-gel medium comprises or defines an elongate path forsaid sample solution, and wherein said one location is intermediateopposite ends of said elongate path.
 3. The method of claim 1 whereinthe dopant metal of said sol-gel is silver.
 4. The method of claim 1wherein the dopant metal of said sol-gel is gold.
 5. The method of claim1 wherein the dopant metal of said sol-gel is copper.
 6. The method ofclaim 1 wherein said sol-gel is hydrophilic.
 7. The method of claim 1wherein said sol-gel is hydrophobic.
 8. The method of claim 1 whereinsaid driving force applied to promote flow of said sample solution intosaid containment means is a vacuum force.
 9. The method of claim 1wherein said driving force applied to promote flow of said samplesolution into said containment means is a pressure force.
 10. The methodof claim 1 wherein said sol-gel medium comprises or defines an elongatepath for said sample solution, and wherein said one location is isolatedbetween opposite ends of said elongate path.
 11. The method of claim 10wherein said elongate path comprises a small bore channel.